Some of native tissues have the orientation of highly organized cells and/or extracellular matrices (ECMs). The orientation of aligned muscle tissues is a key factor for generating a mechanical function in native skeletal muscle having a highly organized structure comprising the parallel fascicles of muscle fibers. In the early stage of development of mature skeletal muscle, myoblasts which are precursor muscle cells are fused to form myotubes, and the newly formed myotubes are merged to be in directions parallel to each other and thereafter finally mature to muscle fibers. Accordingly, control of alignment of myoblasts is an important step for constructing muscle tissue mimicking a living body.
To date, a lot of finely processed materials have been developed for controlling the alignment of myoblasts (Non Patent Literatures 1 to 5). However, success has not been attained in biotissue engineering fields because it has been basically impossible to separate myoblasts from such surfaces subjected to fine processing. Such inseparable coupling has precluded the design of myoblast and myotube structures oriented to three dimensions.
Tissue-like cell monolayers called “cell sheets” have been developed, and the new field of tissue regeneration technology has been established. Poly(N-isopropylacrylamide) (PIPAAm) which is a thermoresponsive polymer grafted on a cell culture substrate has enabled confluent cells to be harvested without being damaged as a single cell sheet at a culture temperature allowed to be lower than 32° C. which is the lower critical solution temperature (LCST) of PIPAAm (Patent Literatures 1 and 2). Such a cell sheet can be transplanted to a damaged tissue without an additional procedure such as suture because the cell sheet associated with an entire ECM can be harvested. A myoblast sheet has been implanted for treating severe heart failure based on the technology (Non Patent Literature 6). Furthermore, biotissue engineering based on such cell sheets has enabled a 3D tissue without a scaffold to be created by layering a plurality of cell sheets (Non Patent Literatures 7 and 8). ECMs held on individual cell sheets enable the layered cell sheets to have definite layer shapes and to physically and biologically interact with each other (Non Patent Literatures 9 and 10).
Patent Literature 3 discloses a sheet-shaped three-dimensional structure for application to heart diseases, comprising myoblasts, and Patent Literature 4 discloses layered cell sheets with myoblasts or the like. In the literatures, however, the orientations of cells in each layer have not been controlled.
Non Patent Literature 7 discloses that vascular endothelial cells sandwiched between myoblast sheets are cultured. In the literature, however, the orientations of cells in each layer have not been controlled.